Proteorhodopsin (pR) is a bacterial light-driven proton pump with several physico-chemical properties similar to those of its archaeal homolog bacteriorhodospin (bR). Currently, there is at least 1 commercial device utilizing bR, a holographic interferometer (the Fringemaker®). The related protein pR is a recently discovered retinal protein with some properties strikingly similar to those of bR, while other properties are different and better suited for certain potential applications. An ability to produce and purify protein inexpensively in large quantities is an important pre-requisite for commercializing pR as a biomaterial. Methods for expressing pR heterologously in E. coli in a functional form have been described previously, but these methods produce pR at a level of <0.1% of total cell dry weight, and require expensive and/or time-consuming column chromatographies to produce pR at a concentration suitable for use in devices.
Technology using biological materials as platforms for developing various electronic and computational devices at the molecular level has advanced greatly in the last decade. The search for biological molecules that can function as durable photochromic materials for information storage and processing is accelerating. Bacteriorhodospin (bR), a light-driven proton pump, mediating photosynthesis in Halobacteria (Oesterhelt, 1989), has long stood out as an important contender for various technical applications in the field of molecular electronics. The ease of preparation of bR in large quantities, coupled with its thermal and photochemical stability, were key factors for investigating its use in holography (Bunkin et al, 1981; Hampp et al, 1988, 1989), optical information processing (Hampp et al, 1990), and other human-invented applications, as well as its natural function of solar energy conversion (Drachev et al, 1976). Genetic engineering has further aided by producing variants of bR with more attractive features (Birge at al, 1990). (For a review of further device applications, see Stuart et al, 2003).
Although the research involving bR as a material for information processing has multiplied enormously, there has been only one commercial product developed where bR is used as a functional component, the Fringemaker® holographic interferometer (Hampp and Juchem, 2000). This calls for exploring newer biological materials with similar properties as bR that might have potential utility in the field of molecular electronics.
Proteorhodopsin (pR), the first-discovered eubacterial homolog of bR, was recently identified from the DNA sequences of several uncultured species of γ-proteobacteria, which are a component of marine planktons present in the ocean surface waters (Béjà et al., 2000, 2001). It is a 249-amino-acid polypeptide with 7 transmembrane α-helices having a retinal co-factor attached to Lys231 to form a protonated Schiff base. When expressed heterologously in E. coli, the pR contained in the bacterial membranes was shown to act as a light-driven proton pump, producing a proton motive force that could be (presumably) be further converted into chemical energy (Béjà et al, 2000). The discovery of a bR homolog in the bacterial kingdom is scientifically significant, since all three kingdoms of life are now known to possess genes for proteins in the bR superfamily. pR is believed to play an important role in the energy balance of the earth due to presence of extensive biomass of marine bacterioplanktons.
In pR, Asp97 and Glu108 almost certainly function respectively as the proton acceptor from, and donor to, the Schiff base (Béjà et al, 2000; Krebs et al, 2002; Dioumaev et al, 2002; Friedrich et al, 2002). These residues are homologous to Asp85 and Asp96 in bR. At pH 9.5 the photocycle of pR was proposed to have pR, pK, pM, pN and pO photointermediate states, quite similar to the bR photocycle, which has six principal photointermediate states: bR, K, L, M, N and O. There is also evidence of a fast (<100 μs) light-triggered proton release from pR at pH>9.0 (Krebs et al. 2002), as also observed in bR. These are just a few of the similarities between pR and bR.
However, one can also find sharp differences between a number of physiochemical attributes of these two proteins. For example, the pKa of the proton acceptor Asp97 in pR in the unphotolyzed state is at least 7.0 (Dioumaev et al. 2002; Friedrich et al. 2002), and in fact for pure protein is closer to 8.2 (Partha et al. 2004). This is 5.5 units more than the pKa (2.5) of the homologous residue in bR, Asp85 (Brown et al. 1993). The spectral maximum of pR is also significantly blue shifted; pR absorbs maximally at 545 nm at pH 7.0 and at 520 nm at pH 9.0, whereas bR absorbs maximally at 570 nm at both these pH values.
Proteorhodopsin has been previously purified using adsorption/affinity chromatography using Phenylsepharose™, hydroxyapatite and/or Ni-NTA resin (Dioumeav et al. 2002; Krebs et al. 2002). To allow purification by using Ni-NTA resin, pR had been cloned into the pBAD-TOPO vector with a 6×His-tag at the C-terminus (Béjà et al. 2000).